Abstract
Protozoal infections caused by species belonging to Leishmania donovani complex are responsible for the most severe form of leishmaniasis, especially in Sudan and other developing countries. Drugs commonly used for the treatment of the disease show varying levels of effectiveness and also have associated side effects. Thus, the present work highlights the synthesis of some chalcones to be used as potential anti-leishmanial agents. The activity of the synthesized chalcones has been evaluated against L. donovani. The ADMET profile of the synthesized compounds were tested using various integrated web-based tools. Moreover, in order to investigate the molecular mechanism of action, the chalcone compounds were docked into L. donovani trypanothione reductase (TR) using Autodock 4.0 and molecular dynamics were studies. Eight compounds showed the highest activity against the morphological forms. Among these compounds, chalcones 15 has shown the highest inhibitory effect with IC50 value of 1.1 µM. In addition, pharmacokinetic and toxicological investigations revealed its good oral bioavailability and low toxicity. Furthermore, chalcone 15 was found to interact with high affinity (−13.7 kcal/mol) with TR, an essential enzyme for the leishmanial parasite. Thus, this promising activity against L. donovani supports the use of chalcone 15 as a potential new therapy for visceral leishmaniasis.
1 Introduction
Leishmaniasis, which is spread by protozoan parasites of the genus Leishmania, is the world’s second most deadly parasitic illness after malaria [1]. It is found in more than 85 countries with those in the tropic and subtropics’ setting being the most affected ones [2–4]. There are three main forms of leishmaniases; visceral, cutaneous, and mucocutaneous [1], where visceral leishmaniasis (VL) is the most life-threatening form of disease which if untreated is fatal [5].
VL is transmitted by the bite of infected L. donovani phlebotomine sand fly female. L. donovani parasite exists in two morphological forms: the amastigote form which is round non-motile, 3–7 µm and the promastigote which is spindle shaped, triple the size of amastigote with flagella [6,7]. The disease is caused by L. donovani complex in East Africa and Indian subcontinent, and by Leishmania infantum in many parts of the world including Latin America and Mediterranean Basin [4,8,9]. VL affects 30,000 people worldwide annually, where 90% occurs in Sudan, Ethiopia, Bangladesh, India, Nepal, and Brazil [3].
Although current available drugs used for VL such as amphotericin B, paromomycin, and miltefosine are not highly effective, not affordable with high cost, and cause serious side effects, they remain the only treatment available for this disease. As a result, there is a pressing need to identify novel compounds of advantage act against the L. donovani parasite and that can outweigh the problem of the available anti-leishmanial drugs [10,11].
The identification of drug target that can reduce the prevalence of Leishmania without inducing rapid resistance, thus remains a major challenge for researcher in the field of infectious disease and medicinal chemist designer [12]. A good starting point for the identification of drug targets is to pinpoint differences between essential metabolic pathways of the host and parasite, which are easier to identify once the parasite physiology and host–parasite relationships are better understood [13].
Unlike the mammals, which rely on glutathione to maintain intracellular redox homeostasis, protozoan parasites of the Trypanosomatidae family depend on a distinct route. Trypanothione pathway, which involves two main enzymes (trypanothione reductase [TR] and synthase [TryS]), is one of the most essential pathways containing novel anti-leishmanial targets. This pathway is responsible for regulating oxidative stress and maintaining the cellular redox potential, so it is essential for parasite survival. In this pathway, the parasite’s thiol-dependent redox polyamine metabolism serves as an important detoxifying system for removing harmful endogenous compounds [14,15]. Because inhibition of TryS and/or TR disrupts the parasite’s redox equilibrium [16,17], these two enzymes have been identified as potential targets for the design of effective anti-leishmanial medicines [15,18,19].
Nature is a never-ending supply of medicinally important chemicals. The use of medicinal plants for parasitic illness therapy has been documented since ancient times [20]. Chalcones (1,3-diphenylpropane 1-one) are diverse group of naturally occurring plant metabolites which have been shown to have a diverse range of biological functions [21–26]. Although, the natural chalcones licochalcone A and isocordoin were found to be efficient in controlling the spread of numerous Leishmania species [27–29], the intrinsic cytotoxicity of these chemicals was a drawback. Thus, the aim of the present study was to synthesize some chalcone compounds targeting a key enzyme in the polyamine-trypanothione pathway, i.e., TR. In vitro and in silico studies for the synthesized compounds were conducted to evaluate their anti-leishmanial activity against L. donovani amastigotes, safety and bioavailability.
2 Materials and methods
2.1 Chemicals
All solvents and reagents used for synthesis were of general reagent grade and were supplied by Sigma-Aldrich. Benzaldehyde, 4-methylbenzaldehyde, 4-methoxybenzaldehyde, naphthaldehyde, 4-hydroxybenzaldehyde, 4-ethylbenzaldehyde, 4-ethoxybenzaldehyde, 2,4-dichlorobenzaldehyde, 4-hydroxy-2-methoxybenzaldehyde, acetophenone, 4-chloroacetophenone, 4-fluoroacetophenone, 4-bromoacetophenone, 4-ethylacetophenone, 4-methylacetophenon, ethanol, methanol, sodium hydroxide, and potassium carbonate.
2.2 Instruments
1H nuclear magnetic resonance (1H NMR), Bruker AN500 instrument; Finnigan LTQ HPLC-MS spectrophotometer with electrospray ionization probe; Thermo Quest CE Elemental analyzer; and thin layer chromatography with Merck precoated silica plates (G F254).
2.3 Synthesis and identification of chalcones, general procedure
An equimolar mixture of substituted acetophenone and substituted benzaldehyde in ethanol (20 mL) were stirred in ice bath, and then 10% of sodium hydroxide (2.5 mL) was added dropwise. Stirring was continued for 30 min to 1 h, depending on the derivative under synthesis. The mixture was kept for overnight in fridge (4°C); the solid substance was filtered off, and washed with ice cold water, followed by cold ethanol or methanol. The product was recrystallized from ethanol or methanol and purified by column chromatography on silica gel (petroleum ether: ethyl acetate, 8:2). Reaction completion was followed using the TLC, and synthesized compounds were identified using NMR, MS, and elemental analysis [30–32].
2.3.1 (E)-1,3-Di-p-tolylprop-2-en-1-one (CH 1)
Yield 96%. 1H NMR (500 MHz, CDCl3) δ 7.91 (d, J = 8.0 Hz, 2H at position 2′, 6′-H), 7.78–7.73 (second-order doublet, J = 8.0 Hz, 1H, 2-H), 7.52 (d, J = 8.0 Hz, 2H, 2″, 4″-H), 7.47 (second order d, J = 15.7 Hz, 1H, 3-H), 7.27 (d, J = 8.0 Hz, 2H, 3′, 5′-H), 7.20 (d, J = 8.0 Hz, 2H, 3″, 5″-H), 2.3× (s, 3H, CH3), 2.4× (s, 3H, CH3). MS (m/z) calculated for C17H16O 236.12 found (M+) 237.17 (100%); Analysis calc. C, 86.4; H, 6.8, found C, 86.5; H, 6.8.
2.3.2 (E)-1-(4-Bromophenyl)-3-p-tolylprop-2-en-1-one (CH 2)
Yield 96%. 1H NMR (500 MHz, CDCl3) δ 7.85 (d, J = 8.0 Hz, 2H at positions 2′, 6′), 7.77 (d, J = 15.0 Hz, 1H at position 2-H), 7.61 (d, J = 8.0 Hz, 2H, 3′, 5′-H), 7.51 (d, J = 8.0 Hz, 2H, 2″, 6″-H), 7.40 ( d, J = 15.0 Hz, 1H, 3-H), 7.21 (d, J = 8.0 Hz, 2H, 3″, 5″-H), 2.73 (s, 3H, CH3).
MS (m/z) calculated for C16H13BrO 300.01 found (M+) 301.11 (100%); Analysis calc. C, 63.8; H, 4.4, found C, 64.0; H, 4.3.
2.3.3 (E)-1-(4-Bromophenyl)-3-(4-ethylphenyl) prop-2-en-1-one (CH 3)
Yield 88%. 1H NMR (500 MHz, CDCl3) δ 7.89 (d, J = 8.0 Hz, 2H, 2′, 6′-H), 7.86 (d, J = 15.0 Hz, 1H, 2-H), 7.69 (d, J = 8.0 Hz, 2H, 3′, 5′-H), 7.60 (d, J = 8.0 Hz, 2H, 2″, 6″-H), 7.47 (d, J = 15.0 Hz, 1H, 3-H), 7.28 (d, J = 8.0 Hz, 2H, 3″, 5″-H), 2.72 (q, J = 8.0 Hz, 2H, CH 2 CH3),1.29 (t, J = 8.0 Hz, 3H, CH2CH 3 ). MS (m/z) calculated for C17H15BrO 314.03 found (M+) 315.00 (100%); Analysis calc. C, 64.8; H, 4.8 found C, 64.9; H, 4.7.
2.3.4 (E)-1-(4-Bromophenyl)-3-(4-hydroxyphenyl) prop-2-en-1-one (CH 4)
Yield 59%. 1H NMR (500 MHz, DMSO) δ 10.15 (s, 1H, OH), δ 8.08 (d, J = 8.0 Hz, 2H, 2′, 6′-H), 7.86 (d, J = 15.0 Hz, 1H, 2-H), 7.75 (d, J = 8.0 Hz, 2H, 3′, 5′-H), 7.60 (d, J = 8.1 Hz, 2H, 2″, 6″-H), 7.47 (d, J = 15.0 Hz, 1H, 3-H), 6.80 (d, J = 8.1 Hz, 2H, 3″, 5″-H). MS (m/z) calculated for C15H11BrO2 301.99 found (M+) 301.24 (100%); Analysis calc. C, 59.4; H, 3.7; found C, 59.3; H, 4.1.
2.3.5 (E)-3-(4-Ethoxyphenyl)-1-(4-ethylphenyl) prop-2-en-1-one (CH 5)
Yield 93%. 1H NMR (500 MHz, CDCl3) δ 7.98 (d, J = 8.1 Hz, 2H, 2′,6′-H), 7.81 (d, J = 15.0 Hz, 1H, 2-H), 7.62 (d, J = 8.1 Hz, 2H, 3′, 5′-H), 7.44 (d, J = 15.0 Hz, 1H, 3H), 7.35 (d, J = 8.0 Hz, 2H, 2″,6″), 6.95 (d, J = 87.9 Hz, 2H, 3″, 5″), 4.11 (q, J = 7.0 Hz, 2H, OCH 2 CH3), 2.76 (q, J = 8.0 Hz, 2H, CH 2 CH3), 1.47 (t, J = 7.0 Hz, 3H, OCH2 CH 3 ), 1.31 (t, J = 8.0 Hz, 3H, CH2 CH 3 ).
MS (m/z) calculated for C19H20O2 280.15 found (M+) 281.20 (100%); Analysis calc. C, 81.4; H, 7.2; found C, 75.3; H, 4.4.
2.3.6 (E)-1-Phenyl-3-Phenyl-prop-2-en-1-one (CH 6)
Yield 88%. 1H NMR (500 MHz, CDCl3) δ 8.08–8.02 (m, 2H), 7.85 (d, J = 15.7 Hz, 1H), 7.70–7.66 (m, 2H), 7.62 (t, J = 7.4 Hz, 1H), 7.55 (d, J = 18.0, 3H), 7.47–7.44 (m, 3H). MS (m/z) calculated for C15H12O 208.09 found (M+) 209.11 (100%); Analysis calc. C, 86.51; H, 5.81; found C, 86.66; H, 5.70.
2.3.7 (E)-1-Phenyl-3-p-tolylprop-2-en-1-one (CH 7)
Yield 92%. 1H NMR (500 MHz, CDCl3) δ 7.99 (m, J = 7.3 Hz, 5H), 7.77 (d, J = 15.0 Hz, 1H at position 2-H), 7.51 (d, J = 8.0 Hz, 2H, 2″, 6″-H), 7.40 (d, J = 15.0 Hz, 1H, 3-H), 7.20 (d, J = 8.0 Hz, 2H, 3″, 5″-H), 2.73 (s, 3H, CH3). Analysis calc. C, 50.63 (100%); H, 4.45; found C, 50.53; H, 4.45. MS (m/z) calculated for C16H14O 222.1 found (M+) 223.17; Analysis calc. C, 86.45; H, 6.37, found C, 83.99; H, 6.07.
2.3.8 (E)-1-p-Tolyl-3-Phenyl-prop-2-en-1-one (CH 8)
Yield 80%. 1H NMR (500 MHz, CDCl3) δ 7.97 (d, J = 8.0 Hz, 2H at positions 2′, 6′), 7.84 (d, J = 15.7 Hz, 1H at position 2-H), 7.67 (d, J = 6.0 Hz, 2H, 3′, 5′-H), 7.57 (d, J = 15.7 Hz, 1H, 3-H), 7.48–7.41 (m, 3H), 7.34 (d, J = 8.0 Hz, 2H), 2.47 (s, 3H, CH3). MS (m/z) calculated for C16H14O 222.1 found (M+) 223.15 (100%); Analysis calc. C, 86.45; H, 6.37, found C, 86.91; H, 6.26.
2.3.9 (E)-1-(4-Methylphenyl)-3-(naphthalen-2-yl) prop-2-en-1-one (CH 9)
Yield 96%. 1H NMR (500 MHz, CDCl3) δ 8.07 (s, 1H), 8.04–7.98 (m, 3H), 7.94–7.82 (m, 4H), 7.69 (d, J = 15.6 Hz, 1H), 7.56 (d, J = 6.0 Hz, 2H), 7.35 (d, J = 7.7 Hz, 2H), 2.48 (s, 3H). MS (m/z) calculated for C20H16O 272.12 found (M+) 273.19 (100%); Analysis calc. C, 88.20; H, 5.92 found C, 87.67; H, 5.79.
2.3.10 (E)-1-(4-Bromophenyl)-3-(4-methoxyphenyl) prop-2-en-1-one (CH 10)
Yield 98%. 1H NMR (500 MHz, CDCl3) δ 7.91 (d, J = 8.0 Hz, 2H at positions 2′, 6′), 7.82 (d, J = 15.6 Hz, 1H at position 2-H), 7.66 (d, J = 8.0 Hz, 2H, 3′, 5′-H), 7.63 (d, J = 8.0 Hz, 2H, 2″, 6″-H), 7.38 (d, J = 15.0 Hz, 1H, 3-H), 6.97 (d, J = 8.7 Hz, 2H, 3″, 5″-H), 3.89 (s, 3H, CH3). MS (m/z) calculated for C16H13BrO2 316.01 found (M+) 317.08 (100%); Analysis calc. C, 60.59; H, 4.13 found C, 60.65; H, 4.00.
2.3.11 (E)-1-(4-Bromophenyl)-3-(4-Ethoxyphenyl) prop-2-en-1-one (CH 11)
Yield 80%. 1H NMR (500 MHz, CDCl3) δ 7.91 (s, J = 8.0 Hz, 2H at positions 2′, 6′), 7.82 (d, J = 15.6 Hz, 1H at position 2-H), 7.66 (d, J = 8.3 Hz, 2H, 3′, 5′-H), 7.62 (d, J = 8.8 Hz, 2H, 2″, 6″-H), 7.38 (d, J = 15.0 Hz, 1H, 3-H), 6.95 (d, J = 8.7 Hz, 2H, 3″, 5″-H), 4.11 (q, J = 7.0 Hz, 2H, CH2), 1.47 (t, J = 7.0 Hz, 3H, CH3). MS (m/z) calculated for C17H15BrO2 330.03 found (M+) 331.10 (100%); Analysis calc. C, 61.65; H, 4.56 found C, 61.63; H, 4.45.
2.3.12 (E)-1-(4, 6-Dimethoxyphenyl)-3-(4, 6-dichlorophenyl) prop-2-en-1-one (CH 12)
Yield 98%. 1H NMR (500 MHz, CDCl3) δ 7.96 (d, J = 12.0 Hz, 1H), 7.85–7.60 (m, 2H), 7.49 (d, J = 20.0 Hz, 1H), 7.40–7.30 (m, 1H), 6.99 (d, J = 7.0 Hz, 1H), 6.65–6.38 (m, 2H), 3.95–3.85 (m, 6H). MS (m/z) calculated for C17H14Cl2O3 336.03 found (M+) 337.11 (100%); Analysis calc. C, 60.55; H, 4.18 found C, 60.29; H, 4.23.
2.3.13 (E)-1-(4-Fluorophenyl)-3-(4-Ethoxyphenyl) prop-2-en-1-one (CH 13)
Yield 93%. 1H NMR (500 MHz, CDCl3) δ 8.10–8.04 (d, 2H at positions 2′, 6′), 7.81 (d, J = 15.0 Hz, 1H at position 2-H), 7.61 (d, J = 6.0 Hz, 2H, 3′, 5′-H), 7.41 (d, J = 15.0 Hz, 1H, 3-H), 7.22 (d, 2H, 2″,6″), 6.95 (d, J = 9.0, Hz, 2H, 3″, 5″-H), 4.12 (q, J = 7.0 Hz, 2H, CH2), 1.47 (t, J = 7.0 Hz, 3H, CH3). MS (m/z) calculated for C17H15FO2 270.11 found (M+) 271.14 (100%); Analysis calc. C, 75.54; H, 5.59; found C, 75.54; H, 5.46.
2.3.14 (2E)-1-(2,4-Dimethoxyphenyl)-3-(4-Fluorophenyl)prop-2-en-1-one (CH 14)
1H NMR (500 MHz, CDCl3) δ 7.79 (d, J = 8.0 Hz, 1H position 6′), 7.67 (d, J = 15.0 Hz, 1H at position 2-H), 7.60 (d, J = 8.6 Hz, 2H, 3″, 5″-H), 7.47 (d, J = 15.0 Hz, 1H, 3-H), 7.10 (d, J = 8.0 Hz, 2H, 2″,6″), 7.00–6.81 (m, 1H, 3′-H), 6.60 (d, J = 8.6, 1H, 5′-H), 3.94 (s, 3H, CH3), 3.90 (s, 3H, CH3). MS (m/z) calculated for C20H15O3 310.16 found (M+) 311.20 (100%); Analysis calc. C, 77.39; H, 7.14; found C, 77.25; H, 7.02.
2.3.15 (E)-1-(4-Fluorophenyl)-3-(4-hydroxy-2-methoxy phenyl) prop-2-en-1-one (CH 15)
Yield 43%. 1H NMR (500 MHz, DMSO) δ 10.18 (s, 1H, OH), 7.99 (d, J = 8.0 Hz, 2H, 2′, 6′-H), 7.95 (s, 1H, 3″-H), 7.80 (d, J = 8.4 Hz, 1H, 2-H), 7.75 (d, J = 8.0 Hz, 2H, 3′, 5′-H), 7.68 (d, J = 15.6 Hz, 1H, 3-H), 7.36 (d, J = 8.0 Hz, 1H, 6″-H), 6.51–6.43 (m, 1H, 5″-H), 2.41 (s, 3H, CH3). MS (m/z) calculated for C17H13O3 268.11 found (M+) 269.14 (100%); Analysis calc. C, 76.10; H, 6.01; found C, 74.69; H, 6.33.
2.3.16 (E)-1-Phenyl-3-(4-methoxyphenyl) prop-2-en-1-one (CH 16)
Yield 82%. 1H NMR (500 MHz, CDCl3) δ 7.78 (d, J = 15.0 Hz, 1H, 2-H), 7.63 (d, 2H, 2″, 6″-H), 7.61–7.57 (m, J = 8.0 Hz, 2H, 3″, 5″-H), 7.36 (d, J = 15.0 Hz, 1H, 3-H), 7.1(m, 3H, 2′, 4′, 6′-H), 6.92 (m, 2H, 3′, 5′-H), 3.84 (s, 3H, CH3). MS (m/z) calculated for C16H14O2 238.10 found (M+) 239.19 (100%); Analysis calc. C, 80.65; H, 5.92; O; found C, 80.75; H, 5.89.
2.3.17 (E)-1-(4-Chlorophenyl)-3-(4-methoxyphenyl) prop-2-en-1-one (CH 17)
Yield 78%. 1H NMR (500 MHz, CDCl3) δ 7.90 (d, J = 19.0 Hz, 2H, 2′, 6′-H), 7.76 (d, J = 15.0 Hz, 1H, 2-H), 7.58 (d, J = 8.0 Hz, 2H, 3′, 5′-H), 7.44 (d, J = 8.0 Hz, 2H, 2″, 6″-H), 7.33 (d, J = 15.0 Hz, 1H, 3-H), 6.92 (d, J = 8.0 Hz, 2H, 3″, 5″-H), 3.84 (s, 3H, CH3). MS (m/z) calculated for C16H13ClO2 272.06 found (M+) 273.17 (100%); Analysis calc. C, 70.46; H, 4.80; found C, 70.61; H, 4.72.
2.3.18 (E)-1-(4-Fluorophenyl)-3-(4-methoxyphenyl) prop-2-en-1-one (CH 18)
Yield 78%. 1H NMR (500 MHz, CDCl3) δ 8.08–7.97 (d, 2H, 2′, 6′-H), 7.78 (d, J = 15.0 Hz, 1H, 2-H), 7.58 (d, J = 8.0 Hz, 2H, 3′, 5′-H), 7.36 (d, J = 15.0 Hz, 1H, 3-H), 7.18–7.08 (d, 2H, 2″, 6″-H), 6.92 (d, J = 8.0 Hz, 2H, 3″, 5″-H), 3.84 (s, 3H, CH3). MS (m/z) calculated for C16H13FO2 256.09 found (M+) 257.17 (100%); Analysis calc. C, 74.99; H, 5.11; found C, 75.3; H, 5.1.
2.3.19 (E)-1-(4-Fluorophenyl)-3-Phenyl-prop-2-en-1-one (CH 19)
Yield 97%. 1H NMR (500 MHz, CDCl3) δ 8.12–8.06 (d, 2H, 2′, 6′-H), 7.85 (d, J = 15.0 Hz, 1H, 2-H), 7.70–7.64 (d, 2H, 3′, 5′-H), 7.53 (d, J = 15.0 Hz, 1H, 3-H), 7.48–7.43 (m, 3H, 2″, 4″,6″-H), 7.24–7.17 (m, 2H, 3″, 5″-H). MS (m/z) calculated for C15H11FO. 226.08 found (M+) 227.12 (100%); Analysis calc. C, 79.63; H, 4.90; found C, 79.78; H, 4.79.
2.3.20 (E)-1-(4-Chlorophenyl)-3-Phenyl-prop-2-en-1-one (CH 20)
Yield 95%. 1H NMR (500 MHz, CDCl3) δ 7.94 (d, J = 8.0 Hz, 2H, 2′, 6′-H), 7.79 (d, J = 15.0 Hz, 1H, 2-H), 7.62 (d, 2H, 3′, 5′-H), 7.53 (d, J = 15.0 Hz, 1H, 3-H), 7.46 (d, J = 12.0 Hz, 3H, 2″, 4″,6″-H), 7.42–7.37 (m, 2H, 3″, 5″-H). MS (m/z) calculated for C15H11ClO 242.05 found (M+) 243.11 (100%); Analysis calc. C, 74.23; H, 4.57; found C, 74.23; H, 4.49.
2.4 In vitro study for intracellular amastigote
2.4.1 Preparation of drugs
Amphotericin B (Ambisome®) was commercially purchased. Stock solutions of synthetic compounds were prepared in culture media for anti-leishmanial assays and then re-sterilized in a laminar flow hood by passing through 0.22 m micro-filters under sterile conditions. In order to make the compounds soluble in water or medium, they were first dissolved in 1% DMSO. All prepared solutions were kept at 4°C and only retrieved when needed.
2.4.2 Intracellular amastigote drug susceptibility assay
Intracellular amastigote susceptibilities assays were determined by infecting THP-1 macrophages (in duplicate) with stationary-phase promastigotes at a ratio of 1:5 for 72 h at 37°C in a 5% CO2–95% air mixture, in 24-well-culture plates. After incubation, non-internalized parasites were removed by washing, and infected macrophage cultures were further incubated at 37°C in a 5% CO2–95% air mixture for 72 h in the absence (control group) or in the presence of standard drugs and different chalcone compounds at several dilutions. At the end of the incubation period, the cells were centrifuged (400×g) for 5 min and the pellets were fixed in 70% analytical-grade methanol (Sigma). Two or three drops of each suspension were transferred onto microscope slides and stained for 5 min in 5% Giemsa. The slides were observed under a light microscope to determine the mean number of amastigotes per/100 macrophages (intensity of infection), and the percent of infected macrophages (rate of infection) [33].
2.5 Statistical analysis of data
Each experiment was performed in triplicates. Results were expressed as mean ± standard deviation of the mean (SD) and analyzed using SPSS 18. The IC50 values were calculated using GraphPad Prism 6.0 software, considering P < 0.05 as significant.
2.6 Homology modeling
2.6.1 Model generation
The protein 3D structure was built via comparative modeling method using the SWISS MODEL server facilities. Using the BLAST feature of SWISS-MODEL, a total of 74 templates were found, with 98% identity to the template sequence. For each identified template, the template’s quality has been predicted from features of the target-template alignment. The template with the highest quality has then been selected for model building. Models were built based on the target-template alignment using ProMod3 [34], and the geometry of the resulting model is then regularized by using a force field. Ligands present in the template structure were transferred by homology to the model.
2.6.2 Model validation
The global and per-residue model quality has been assessed using the QMEAN scoring function [35] and Global Model Quality Estimation. The 3D superposition of the target protein and the template was carried out, and the root mean square deviation (RMSD) for alignment was determined. Additionally, the quality of the protein was assessed employing PROCHECK server by Ramachandran plot [36].
2.7 In silico studies
2.7.1 In silico absorption, distribution, metabolism, and excretion (ADME) properties, toxicity risks assessment, and drug likeliness
SwissADME web tool (http://www.swissadme.ch/) [37] and OSIRIS Property Explorer open-source program (http://www.organicchemistry.org/prog/peo/) were used to assess the ADME properties and toxicity risks of the synthesized chalcones.
2.7.2 Molecular docking and visualization
Molecular docking was performed using AutoDock 4.0 software, based on the Lamarckian genetic algorithm [38,39]. DS Visualizer Client (Windows 64 bit) (267 MB) was then applied to visualize the docking files that showed the lowest binding energies.
2.7.3 Molecular dynamics (MD) simulation
Following molecular docking, MD simulations were performed on two systems: uncomplexed TR and TR complexed with CH 15. MD simulation was utilized to investigate the binding stability of the final complexes using WEBGRO for macromolecular simulations (https://simlab.uams.edu/). Using GROMOS96 43a1 force field parameters, the whole system was solvated in water, neutralized, and 0.15 M salt of NaCl was added. The sharpest descending strategy resulted in an energy decrease of 5,000 steps. The kinds of equilibration employed were constant quantity, volume, temperature (NVT/NPT), and pressure. The temperature was set to 300 K and the pressure was set to 1.0 bar for a 100 ns simulation time and 1,000 frames each simulation. RMSD and root mean square fluctuation (RMSF) were the simulation parameters required. The requested simulation parameters were RMSD, RMSF, radius of gyration (Rg), intermolecular H-bonding (H-bonds), and solvent accessible surface area. Topology file of the protein–ligand complexes was created using PRODRG sever [40].
3 Results
3.1 Chalcone’s derivatives synthesis
Claisen–Schmidt reaction (Scheme 1) was utilized for the synthesis of the chalcones derivatives. The reaction procedure involved the stirring of substituted acetophenones and substituted benzaldehydes at room temperature in ethanol, using 10% NaOH as catalyst. When the benzene rings of the starting acetophenones or benzaldehydes bearing at least one hydroxyl group, the synthetic procedure needed to be modified due to the formation of the phenoxide anion before or during the reaction. The phenoxide ion is produced because sodium hydroxide abstracts the hydrogen of the phenolic hydroxyl, resulting in the negatively charged phenoxide ion, which is stabilized through the formation of resonance hybrids and leads to slowing down the reaction dramatically. For this reason sodium hydroxide was added in excess (double the normal amount) and the reaction was continued – in some cases – for 3 days. Eventually, to obtain the product, reaction mixture was worked up with 1 M aqueous hydrochloric acid to neutralize excess sodium hydroxide in order to convert back the phenolate ion into its original phenolic form. All compounds were then purified by recrystallization from ethanol or methanol. Column chromatography (petroleum ether: ethylacetate, 8:2) was used when recrystallization failed to afford the pure compounds.
Synthesis of chalcone analogues.
In order to perform a structure activity relationship (SAR) study, the two phenyl rings were functionalized with different moieties such as F, Cl, Br, OCH3, OCH2CH3, and OH in different positions (Supplementary File).
4 Intracellular amastigote drug susceptibility assay
4.1 General screening
Twenty synthesized chalcone derivatives at concentration 30 µM were screened against intracellular amastigote using THP1, to calculate the percent of inhibition of each compound separately (Supplementary File). The eight compounds that showed highest percent of inhibition were selected further for the IC50 assay. The IC50 of +ve control (amphotericin B) and the selected chalcones (chalcone 10–15, 17, 18) were calculated using graphBad prism 6.0 (Table 1, Figure 1).
Intracellular amastigote activity of amphotericin B and chalcones compounds with corresponding IC50
| Mean % of inhibition ± SD | |||||||||
|---|---|---|---|---|---|---|---|---|---|
| Conc. (µM) | Amph. B | CH 10 | CH 11 | CH 12 | CH 13 | CH 14 | CH 15 | CH 17 | CH 18 |
| 30 | 75.3 ± 0.58 | 58.7 ± 0.58 | 70.5 ± 0.5 | 71.6 ± 0.53 | 71.6 ± 0.53 | 67.4 ± 0.35 | 73.3 ± 0.46 | 50.3 ± 0.5 | 58.6 ± 0.58 |
| 10 | 72.8 ± 0.68 | 56.8 ± 0.28 | 67.7 ± 0.64 | 69.1 ± 0.17 | 68.3 ± 0.58 | 64.1 ± 0.23 | 69.4 ± 0.75 | 47.6 ± 0.5 | 51.6 ± 0.51 |
| 3 | 67.7 ± 0.58 | 41.2 ± 0.29 | 60 ± 0.00 | 62.7 ± 0.58 | 55.4 ± 0.56 | 59.1 ± 0.17 | 68.9 ± 0.85 | 25 ± 0.00 | 45.1 ± 0.12 |
| 1 | 57.3 ± 0.58 | 36.8 ± 0.29 | 51.2 ± 0.29 | 55 ± 0.00 | 51.8 ± 0.76 | 53.5 ± 0.50 | 62.0 ± 0.55 | 20.3 ± 0.5 | 35.3 ± 0.58 |
| 0.3 | 52.8 ± 0.76 | 33.1 ± 0.23 | 49.3 ± 0.58 | 50.8 ± 0.76 | 41.9 ± 1.01 | 49.3 ± 0.29 | 55.9 ± 0.90 | 17.2 ± 0.68 | 30.6 ± 1.15 |
| IC50 (µM)* | 1.7 | 3.8 | 2.46 | 2.07 | 2.48 | 2.2 | 1.1 | 4.2 | 3.6 |
*IC50 is the anti-logarithm of the concentration at which the curve passes through the 50% of inhibition.
Percent of inhibition of L. donovani amastigote in different concentrations of amphotericin B and chalcone 15 (lowest IC50).
4.2 Homology modeling
The tertiary structure of L. donovani’s TR enzyme was constructed using SWISS-Model server (Figure 2a), while the PROCHECK server’s Ramachandran plot revealed that 90.6% of the residues are situated in the most favorable area (Figure 2b). QMEAN was also used to check the quality of the projected 3D structure (Figure 3). The Z-score for QMEAN analysis was −1.73, and the overall score was 0.718. These values indicate that the model is of better quality, with an acceptable score ranging from 0 to 1 [36].
(a) 3D modeled structure of L. donovani TR and (b) Ramachandran plot analysis to validate the 3D predicted structure.
Assessment of structural superiority by QMEAN valuation.
4.3 In silico studies
4.3.1 In silico ADME properties
The predicted results for the drug likeliness and physicochemical properties of the most potent compounds are summarized in Tables 2 and 3.
Physicochemical properties of the synthesized compounds
| Compound No. | MW (g/mol) | NRBs | NHBAs | NHBDs | TPSA (A°2) | Consensus log P(o/w) | Water solubility class |
|---|---|---|---|---|---|---|---|
| CH 10 | 317.18 | 4 | 2 | 0 | 26.3 | 4.06 | Moderately soluble |
| CH 11 | 331.2 | 5 | 2 | 0 | 26.3 | 4.41 | Poorly soluble |
| CH 12 | 337.2 | 5 | 3 | 0 | 35.53 | 4.40 | Poorly soluble |
| CH 13 | 270.3 | 5 | 3 | 0 | 26.3 | 4.41 | Moderately soluble |
| CH 14 | 286.3 | 5 | 4 | 0 | 35.53 | 3.66 | Moderately soluble |
| CH 15 | 272.27 | 4 | 4 | 1 | 46.53 | 3.27 | Soluble |
| CH 17 | 272.73 | 4 | 2 | 0 | 26.3 | 3.97 | Moderately soluble |
| CH 18 | 256.27 | 4 | 3 | 0 | 26.3 | 3.97 | Moderately soluble |
Predicted ADME properties of the synthesized compounds
| Molecule No. | HIA | BBB permeant | Pgp substrate | CYP1A2 inhibitor | CYP2C19 inhibitor | CYP2C9 inhibitor | CYP2D6 inhibitor | CYP3A4 inhibitor | log Kp (cm/s) |
|---|---|---|---|---|---|---|---|---|---|
| CH 10 | High | Yes | No | Yes | Yes | Yes | Yes | No | −4.97 |
| CH 11 | High | Yes | No | Yes | Yes | Yes | Yes | No | −4.71 |
| CH 12 | High | Yes | No | Yes | Yes | Yes | Yes | Yes | −4.89 |
| CH 13 | High | Yes | No | Yes | Yes | No | Yes | No | −4.76 |
| CH 14 | High | Yes | No | Yes | Yes | No | Yes | Yes | −5.4 |
| CH 15 | High | Yes | No | Yes | Yes | No | No | Yes | −5.54 |
| CH 17 | High | Yes | No | Yes | Yes | Yes | Yes | No | −4.74 |
| CH 18 | High | Yes | No | Yes | Yes | No | Yes | No | −5.02 |
4.3.2 Toxicity risks and drug likeliness
The eight chalcones were tested for their risk toxicity and drug likeliness. Only five compounds were predicted to have no risk of mutagenic, tumorigenic, irritant, or reproductive effects as shown in Table 4.
Toxicity risks and drug likeliness predicted by OSIRIS Property Explorer
| Compound | ME | TE | IE | RE | DL | DS |
|---|---|---|---|---|---|---|
| CH 10 | − | − | +++ | ++ | −1.86 | 0.18 |
| CH 11 | − | − | − | − | −2.92 | 0.31 |
| CH 12 | − | − | − | − | 0.52 | 0.45 |
| CH 13 | − | − | − | − | 0.59 | 0.58 |
| CH 14 | − | − | − | − | −1.5 | 0.45 |
| CH 15 | − | − | − | − | 1.48 | 0.73 |
| CH 17 | − | − | +++ | ++ | 3.23 | 0.32 |
| CH 18 | − | − | +++ | ++ | 1.69 | 0.34 |
ME, mutagenic effect; TE, tumorigenic effect; IE, irritant effect; RE, reproductive effect; DL, drug likeliness; DS, drug score; (+++): high risk, (++): medium risk, and (−): no risk.
4.3.3 Molecular docking
The molecular docking was conducted for the most promising compound (chalcone 15) in terms of bioavailability, safety, and anti-leishmanial activity (Figure 4).
Molecular docking of Chacone 15 bound in the active site of T.
4.3.4 MMD simulation
The MD results for the stability and flexibility of CH 15-TR complex are shown in Figures 5 and 6.
The RMSD profiles of protein backbone (a) and for CH 15-TR complex (b).
The RMSF profile of CH 15-TR complex.
5 Discussion
To help meet the critical need for safe and effective anti-leishmaniasis drugs. Several bioactive compounds with anti-leishmanial activity and diverse modes of action have been synthetized [41,42]. A variety of natural and synthetic chalcones have been shown to have anti-leishmanial action [28,43–46]. Another reports described the activity of different chalcone derivatives against various targets of leishmania [47–49]. However, the present report describes more effective, safer chalones derivatives for the inhibition of TR enzyme of L. donovani, employing amphotericin B as reference compound.
5.1 In vitro anti-leishmanial amastigote activity and SAR
Following the successful synthesis of the designed chalcones, their anti-leishmanial activities were then assessed as percentage of inhibition and 50% inhibitory concentration (IC50). The percent of inhibition of all 20 screened compounds was found ranging between 73 and 37%, with only eight analogues showing the highest inhibition effect and selected for IC50 assay which was conducted at dose range of 30–0.3 µM (Table 1). It is more likely that, the different substitutions on rings A and B of chalcone scaffold play a pivotal role on the biological activity of these compounds. The selected chalcones showed significant IC50 values (P ≤ 0.05) in the range 1.1–4.2 µM when compared to amphotericin B whose IC50 value was 1.7 μM (Table 1).
According to the IC50 results, among the halide substituted chalcones at 1-phenyl moiety or 2-phenyl moiety the fluorine atom appears to have the best role in the amastigote inhibitory activity, where the IC50 of chalcone 18 was 3.6 ± 0.67 µM, compared to 3.8 ± 0.35 and 4.2 ± 0.54 µM for chalcones 10 and 17 having Br and Cl atoms, respectively. It was also observed that besides the presence of halide atom, chalcones having ethoxy substitution at 1-phenyl moiety as chalcones 11 and 13 (IC50 2.46 ± 0.46 and 2.48 ± 0.71, respectively) exhibit better inhibitory activity than those chalcones having methoxy substituents (chalcones 10, 17, 18 with IC50 3.8 ± 0.35, 4.2 ± 0.54, and 3.6 ± 0.67 µM, respectively). Moreover, the length and number of alkoxy substitution proved to have great effect on the inhibitory activity (chalcones 11, 14).
Interestingly, the most potent effect with highest inhibition activity was obtained when one of the methoxy group was replaced by hydroxyl group at 1-phenyl moiety as chalcone 15. Chalcone 15, with IC50 1.1 ± 0.72 µM and 73% inhibition, is proved to be significantly more potent than amphotericin B with IC50 1.7 ± 0.63.
5.2 ADME properties and toxicity risk
Pharmacokinetic and toxicological characteristics are critical components of drug design in order to obtain excellent oral bioavailability and safe medicines. As a result, ADMET computational assessments were conducted to evaluate the eight chalcone derivatives with the highest inhibitory efficacy against TR. Based on the Lipinski rule of 5 [50], our findings imply that these compounds have varied oral bioavailability. Among all of them, chalcone 15 was found to be the only compound having less likely solubility problems (Table 2). In addition, it showed varied inhibitory effect against the different cytochromes (Table 3), which may result in drug–drug interactions [51]. Moreover, with drug scores (DS) >0.5, it also demonstrated no danger of mutagenic, tumorigenic, reproductive, or irritating effects (Table 4). These in silico findings support de Mello et al. results that chalcones with anti-leishmania efficacy have favorable pharmacokinetic and toxicological profiles [52].
5.3 Docking studies
To gain further insights into the mechanism of inhibition, the most active chalcone 15 was docked into the active site of TR. TR’s structure showed two critical cysteine residues in the active site (Cys52 and Cys57), which are engaged in a coordinated nucleophilic attack on the TS2 disulfide bridge to generate the reduced substrate T(SH)2. The most likely and energetically favorable binding mechanisms of compound 15 with TR are depicted in Figure 4. The binding mode showed an estimated binding energy of −13.7 kcal/mol. The docking results revealed that chalcone 15 interacts with the enzyme active site through two hydrogen bonds between the oxygen atom of the compound’s carbonyl group and the residue THR335, and between the oxygen atom of compound’s methoxy group and residue CYS 57. Moreover, different pi- and alkyl bonding were formed between the phenyl group of the compound and residues LYS60, TYR198, ILE 199, ASP 327, and ALA 338.
To further analyze the dynamic atomic interactions of chalcone 15 within the active site, 100 ns TR and its complexes are simulated using WebGro. MD simulation is a robustly accurate strategy to analyze the configurational changes that occur when a ligand is induced to fit. Using WebGro, the protein/complex system is computationally evolved using classical mechanics for a short 100 ns timespan, and the configurational stability or binding affinity of a ligand is assessed across the simulation trajectory. To analyze the simulation results, Rg or the average distance between the center of mass and the rotational axis is usually used to estimate the conformational stability of a system against any physicochemical strain, and its lower score implies a higher stability. Here, as shown in Figure 5, the RMSD plot of the CH 15 bound TR complex initially showed very low variations till 20 ns ranging 0.3–0.4 nm followed by stable conformation till 100 ns. This stability could be attributed to the higher number of stable bonding between compound and the target protein.
Afterwards, the RMSF value was calculated to investigate the structural flexibility of protein’s backbone atoms (Figure 6). Obtained results indicated no high fluctuation and the flexibility of the complex (RMSF ≤ 0.4 nm).
It is clear that there was a great consistency between the biological activity and the docking results. Chalcone 15 was revealed to have good binding with TR enzyme along with having the lowest IC50 1.1 ± 0.72 µM, this could be attributed to the unique substitution on ring B of this compound (OH). Additionally, the methoxy (OCH3) and hydroxyl (OH) group at carbon 1 and 3 of the chalcone skeleton allows hydrophobic and hydrogen bonds interactions with the enzyme.
However, this study has some limitations, including the lack of in vivo pharmacokinetics and pharmacodynamics testing of the selected compounds.
6 Conclusion
Herein, 20 new chalcone derivatives have been designed and synthesized to inhibit L. donovani TR. Among the tested compounds, CH 15 showed inhibitory effect against the parasite’s amastigote higher than the reference inhibitor amphotericin B. Furthermore, the chalcone derivatives were found to have a direct contact with TR’s active site residues, as well as minimal toxicity and good oral bioavailability, according to the in silico studies. This study has also supported the SARs, highlighting the importance of hydrophobic interactions, besides the hydrogen bonds between the compounds and the target under study. The data obtained from the docking-based design of compounds with varying scaffolds gave remarkable information about the type of substituents that can be placed in different positions in ring (A) and (B) of chalcones to improve the binding capacity of the resulting analogues to L. donovani targets. To conclude, the promising activity against L. donovani, compared to amphotericin B and other reported chalcones derivatives, support the use of chalcones 15 as a potential new therapy for VL.
Acknowledgments
The authors are thankful for Ahfad Biomedical Research Center, Ahfad University for Women, Sudan and Institute of Endemic Disease, University of Khartoum, Sudan for their support in conducting the in vitro and intracellular assay. The authors are also grateful for the Researchers Supporting Project number (RSP-2021/119), King Saud University, Riyadh, Saudi Arabia for funding and technical support.
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Funding information: Researchers Supporting Project number (RSP-2021/119), King Saud University, Riyadh, Saudi Arabia.
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Author contributions: M.O. conducted the in vitro and intracellular assay, the SAR study and homology modeling, and wrote the paper draft; T.A. synthesized the compounds and conducted the homology modeling; E.G. conducted part of the SAR study and revised the paper draft; S.S. conducted the in silico studies, homology modeling, and wrote and edited the draft paper; M.M. supervised the intracellular assay. W.O. revised the paper draft; R.M. funded the study and revised the draft; R.I. revised the draft. All authors approved the final draft. All the authors approved the final article.
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Conflict of interest: The authors declare no conflict of interest.
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Data availability statement: All dataset supporting this article is available within the article.
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Ethical approval: The conducted research is not related to either human or animal use.
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